In this study, we have analyzed the interdependence between the polymerase and RNase H active sites of human immunodeficiency virus-1 reverse transcriptase (RT) using an in vitro system that closely mimics the initiation of (؉)-strand DNA synthesis. Time course experiments show that RT pauses after addition of the 12th DNA residue, and at this stage the RNase H activity starts to cleave the RNA primer from newly synthesized DNA. Comparison of cleavage profiles obtained with 3-and 5-end-labeled primer strands indicates that RT now translocates in the opposite direction, i.e. in the 5 direction of the RNA strand. DNA synthesis resumes again in the 3 direction, after the RNA-DNA junction was efficiently cleaved. Moreover, we further characterized complexes generated before, during, and after position ؉12, by treating these with Fe 2؉ to localize the RNase H active site on the DNA template. Initially, when RT binds the RNA/DNA substrate, oxidative strand breaks were seen at a distance of 18 base pairs upstream from the primer terminus, whereas 17 base pairs were observed at later stages when the enzyme binds more and more DNA/DNA. These data show that the initiation of (؉)-strand synthesis is accompanied by a conformational change of the polymerase-competent complex. Retroviral RTs1 are multifunctional enzymes possessing RNA-and DNA-dependent polymerase activities and a ribonuclease H (RNase H) activity that degrades the RNA strand of RNA/DNA hybrids (1, 2). Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) uses a cellular tRNA primer to initiate reverse transcription from a complementary primer-binding site (PBS) near the 5Ј-end of the viral RNA (3-6). Despite changes of binding and kinetic properties, observed concomitant with synthesis of the first DNA strand (7), i.e. (Ϫ)-strand DNA, complexes with the initially bound RNA/ RNA duplex and the newly synthesized DNA/RNA substrates share certain common features. RNase H cleavages on the RNA strand of DNA/RNA primer/template combinations occur at a constant distance of 18 bp upstream of the nascent primer terminus (8, 9). Analogously, RNase H-induced cleavages within the tRNA/RNA duplex, designated as RNase H* activity (10), were observed at the same distance from the 3Ј-end of the primer, although these cuts are restricted to stalled complexes (11). Together, these data provide strong evidence that RT binds to both RNA/RNA and DNA/RNA substrates with the same orientation, and the number of bp between the two active sites is 18 in each case.RT-DNA/DNA complexes, which are generated during (ϩ)-strand synthesis, have been relatively well characterized (12-15). The crystal structure of HIV-1 RT complexed to an 18-base primer/19-base template DNA homoduplex (12) suggests that the first 7 DNA/DNA base pairs near the polymerase active site adopt an A-type conformation, whereas the region further upstream is in the preferred B-conformation, both structurally distinct segments being separated by a kink.Little information is currently available regarding th...
During initiation of minus-strand synthesis by HIV-1 reverse transcriptase, a 3'-DNA-RNA-5' junction is formed involving the 3'-end of tRNAlys,3. The HIV-RT-associated RNase H cleaves the RNA template strand specifically, opposite the newly synthesized DNA strand. We have determined the crystal structure at 1.9 A resolution of an eight-base pair hybrid duplex representing the junction to identify global or local structural perturbations which may be recognized by HIV-RT RNase H. The junction octamer is in a global A-type conformation throughout. A base pair step with distinct stacking geometry and variable backbone conformation is located next to the main endonucleolytic cleavage site. This base pair step may serve as a recognition site for HIV-RT RNase H.
The interaction and subsequent photoaddition reaction on UVA irradiation of 4,4′-dimethyl-5-methoxyangelicin and 4,4′-dimethyl-5-methoxypsoralen to twelve synthetic oligonucleotides was studied. A new approach was developed which allows: (a) accurate determination of the kinetics constant of the overall photoaddition process to each oligonucleotide; (b) identification of the different types of intercalation sites, individualized by a combination of two consecutive base pairs and the frequency of their appearance along the oligomer sequences; and (c) the value of the kinetics constant relative to each type of site to be determined. This approach also makes it possible to highlight the influence of sequences flanking each site that participate in the photoaddition process. Thus, the possibility of discriminating which sites are active in the photoaddition reaction and their contribution to the overall process appears to be a useful tool for studying the correlation between furocoumarin photocombination with DNA and the consequent biological effects.
In order to further investigate the potential of rifamycins as antiinflammatory drugs, twenty-five semisynthetic rifamycins were tested at concentrations ranging from 10(-9) to 10(-5) M on in vitro human neutrophil functions such as locomotion, superoxide anion production, and degranulation, under different stimulatory conditions. They were also tested as antiproliferative agents on peripheral blood lymphocytes. The present semisynthetic derivatives are in general characterized by their carrying a hydrophilic substituent; they are rifamycin S or rifamycin SV derivatives carrying at C(3) either a carboxyalkyl side-chain or a glycosyl side-chain. Derivatives of the former group displayed inhibitory activities covering the whole range of activities tested, suggesting that the sum of these different effects could support their antiinflammatory activity in vivo. These derivatives, carrying a free carboxyl, are more water soluble than rifamycin SV at physiological pH, and may serve as antiinflammatory drugs for local administration, alternative to rifamycin SV, possibly giving higher efficacy and reduced side effects of pain and tissue swelling.
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